Role of Mycobacterium Tuberculosis RecG Helicase in DNA Repair, Recombination and in Remodelling of Stalled Replication Forks
Thakur, Roshan Singh
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Tuberculosis, caused by the infection with Mycobacterium tuberculosis remained as a major global health challenge with one third of world population being infected by this pathogen. M. tuberculosis can persist for decades in infected individuals in the latent state as an asymptomatic disease and can emerge to cause active disease at a later stage. Thus, pathways and the mechanisms that are involved in the maintenance of genome integrity appear to be important for M. tuberculosis survival, persistence and pathogenesis. Helicases are ubiquitous enzymes known to play a key role in DNA replication, repair and recombination. However, role of helicases in providing selective advantage for M. tuberculosis survival and genome maintenance is obscure. Therefore, understanding the role of various helicases could provide insights into the M. tuberculosis survival, persistence and pathogenesis in humans. This information could be useful in considering helicases as a novel therapeutic target as well as developing effective vaccines. The research focus of my thesis has been to understand the role of helicases in safeguarding the M. tuberculosis genome from various genotoxic stresses. The major focus of the current study has been addressed towards understanding the role of M. tuberculosis RecG (MtRecG) helicase in recombinational repair and in remodeling stalled replication forks. This study highlights the importance of RecG helicase in the maintenance of genome integrity via DNA repair, recombination and in remodeling the stalled replication forks in M. tuberculosis. The thesis has been divided into following sections as follows: Chapter I: General introduction that describes the causes and consequences of replication stress and DNA repair pathways in M. tuberculosis The genome is susceptible to various types of damage induced by exogenous as well as endogenous DNA damaging agents. Unrepaired or misrepaired DNA lesions can lead to gross chromosomal rearrangements and ultimately cell death. Thus, organisms have evolved with efficient DNA damage response machinery to cope up with deleterious effects of genotoxic agents. Accurate transmission of genetic information requires error-free duplication of chromosomal DNA during every round of cell division. Defects associated with replication are considered as a major source of genome instability in all organisms. Normal DNA replication is hampered when the fork encounters road blocks that have the potential to stall or collapse a replication fork. The types of lesions that potentially block replication fork include lesions on the template DNA, various secondary structures, R-loops, or DNA bound proteins. To understand the DNA damage induced replication stress and the role of fork remodeling enzymes in the repair of stalled replication forks and its restart, chapter I of the thesis has been distributed into multiple sections as follows: Briefly, initial portion of the chapter describes overall replication process in prokaryotes highlighting the importance of coordinated replisome assembly and disassembly during initiation and termination. Later section discusses about various types of exogenous and endogenous DNA damages leading to replication fork stalling. Subsequent section of chapter I provide detailed description and mechanism of various repair pathways cell operates to repair such damages. Chapter I further summarizes causes of stalled replication forks majorly including template lesions, natural impediments like DNA secondary structures and DNA-protein cross links. Subsequent section discusses various pathways of replication restart that include essential role of primosomal proteins in reloading replisome machinery at stalled replication forks. Subsequent section of chapter I provide a comprehensive description of replication fork reversal (RFR) and mechanism of replication restart. RFR involves unwinding of blocked forks via simultaneous unwinding and annealing of parental and daughter strands to generate Holliday junction (HJ) intermediate. Genetic and biochemical studies highlighted the importance of RecG, RuvAB and RecA proteins in driving RFR reaction in E. coli. Hence, in the subsequent chapter, the functional role of RecG, RuvAB and RecA in replication-recombination processes has been discussed. Last section of the chapter devotes completely to M. tuberculosis, its genome dynamics and the various pathways of mycobacterial DNA repair. M. tuberculosis experiences substantial DNA damage inside host macrophages owing to the acidic environment, reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) which are sufficient enough to cause replication stress. To gain insights into the role of M. tuberculosis RecG helicase in DNA repair, recombination and in remodeling the stalled replication forks the following objectives were laid for my PhD thesis: 1 To understand the functional role of M. tuberculosis RecG (MtRecG) in DNA repair and recombination. 2 To investigate the distinct role(s) of MtRecG, MtRuvAB and MtRecA in remodeling the stalled replication forks. Chapter II: Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination In order to survive and replicate in a variety of stressful conditions during its life cycle, M. tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. Helicases are one such ubiquitous enzyme involved in all DNA metabolic transaction pathways for maintenance of genome stability. To understand the role of M. tuberculosis RecG (MtRecG) helicase in recombination and repair, we carried out functional and biochemical studies. In our study, we show that M. tuberculosis RecG expression was induced in response to different genotoxic agents. Strikingly, expression of M. tuberculosis RecG in Escherichia coli ∆recG mutant strain provided protection against MMC, MMS and UV-induced cell death. Purified M. tuberculosis RecG exhibited higher binding affinity for the Holliday junction (HJ) as compared to a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile junction, branch migration and resolution was evident only in the case of the mobile junction. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently as compared to the mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structures, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Altogether, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as in stress conditions. Chapter III: Mycobacterium tuberculosis RecG but not RuvAB or RecA is efficient at remodeling the stalled replication forks: Implications for multiple mechanisms of replication restart in mycobacteria Aberrant DNA replication, defects in the protection and restart of stalled replication forks are a major cause of genome instability in all organisms. Replication fork reversal is emerging as an evolutionarily conserved physiological response for restart of stalled forks. Escherichia coli RecG, RuvAB and RecA proteins have been shown to reverse the model replication fork structures in vitro. However, the pathways and the mechanisms by which Mycobacterium tuberculosis, a slow growing human pathogen responds to different types of replication stress and DNA damage is unclear. In our study, we show that M. tuberculosis RecG rescues E. coli ∆recG cells from replicative stress. The purified M. tuberculosis RecG (MtRecG) and RuvAB (MtRuvAB) proteins catalyze fork reversal of model replication fork structures with and without leading strand ssDNA gap. Interestingly, SSB suppresses the MtRecG and MtRuvAB mediated fork reversal with substrates that contain lagging strand gap. Notably, our comparative studies with fork structures containing template damage and template switching mechanism of lesion bypass reveal that MtRecG but not MtRuvAB or MtRecA is proficient in driving the fork reversal. Finally, unlike MtRuvAB, we find that MtRecG drives efficient reversal of forks when fork structures are tightly bound by protein. These results provide direct evidence and valuable insights into the underlying mechanism of MtRecG catalyzed replication fork remodeling and restart pathways in vivo.
- Biochemistry (BC) 
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